Proteomic analysis of antioxidant effects exhibited by phikud navakot extract in human umbilical vein endothelial cells under homocysteine treatment

Abstract

Phikud Navakot is a main ingredient in Yahom Navakot, Thai traditional polyherbal formula that has been widely used for treatment of circulatory disorder. In this study, the effect of Phikud Navakot ethanolic extract (NVK) was investigated on homocysteine-induced oxidative stress in human umbilical vein endothelial EA.hy926 cells. Scavenging activities against radicals of superoxide, hydroxyl, nitric oxide and hydrogen peroxide were performed. The results showed that NVK exhibited higher antioxidant activity than most of individual herbal extracts. Pre-treatment of NVK at the non-toxic concentration of 0.05 mg/mL for 12 h prior exposure to 0.05 mM homocysteine (Hcy) significantly reduced intracellular reactive oxygen species. Two-dimensional electrophoresis coupled with mass spectrometry showed that NVK alone altered the protein expression involving in redox regulation, metabolism and chaperones. Hcy altered the level of proteins involving in endothelial dysfunction. Meanwhile, pre-treatment of NVK before exposure to Hcy restored the proteins such as annexin A2, heat shock cognate 71 kD. Network analysis using bioinformatic tools such as STRING, KOBAS, and IPA® revealed signaling pathways affected by NVK in the absence or presence of Hcy. Additionally, immunoblot analysis showed that NVK up-regulated heme oxygenase-1 and vascular endothelial growth factor and activated endothelial nitric oxide synthase. Our investigation provides an understanding in mechanism of action of NVK, supporting the traditional use for the treatment of circulatory disorder.